Serena De Santis

Novel thermosensitive calcium alginate microspheres: Physico-chemical characterization and delivery properties

The system described in this paper was obtained by soaking calcium alginate (CaAlg) microspheres in a water solution of poly-[(3-acrylamidopropyl)-trimethylammonium chloride-b-N-isopropylacrylamide] [poly(AMPTMA-b-NIPAAM)], a new block co-polymer recently synthesized by atom transfer radical polymerization (ATRP). The block co-polymer is characterized by a lower critical solution temperature (LCST) of 41 degrees C in aqueous 0.1 M NaCl solution, and can be anchored on the CaAlg microspheres by means of polyion interactions. Polycations (permanently positively charged blocks) and polyanions (free alginate carboxylic groups) interact, leading to microspheres with thermosensitive properties. As an effect of interaction with the microspheres the LCST of the co-polymer is lowered to 36-38 degrees C. In this temperature range a colloidal water suspension of the microspheres collapses, forming macroscopic aggregates. The new system shows, at human body temperature, an improved ability to carry and deliver both hydrophobic and hydrophilic molecules in comparison with unmodified CaAlg microspheres. The release properties of the microspheres loaded with different model drugs can be appropriately modulated by the amount of the poly(AMPTMA-b-NIPAAM). Furthermore, the microspheres show the interesting capability of retaining the activity of a loaded enzyme (horseradish peroxidase), used as a model protein. The results obtained indicate that the proposed drug delivery system may be suitable for drug depot applications.

Dielectric Properties of Micellar Aggregates Due to the Self-Assembly of Thermoresponsive Diblock Copolymers

The radiowave dielectric properties of aqueous solutions of thermosensitive copolymers, consisting of poly(2-acrylamido-2-methylpropanesulfonate) [PAMPS] and poly(N-isopropylacrylamide) [PNIPAAM] with different block lengths, have been investigated over a broad temperature and frequency range. These copolymers PAMPS(n)-b-PNIPAAM(m) form temperature responsive aggregates (micelles) that represent a class of self-assembled structures in water of great interest because of their potential use as drug delivery formulations and in diverse biotechnological applications. Copolymers formed by hydrophilic segments covalently attached to a hydrophobic segments are capable of forming a micellar structure as soon as the temperature is raised above their lower critical solution temperature. We have investigated the dielectric properties of PAMPS(n)-b-PNIPAAM(m) diblock copolymers with different lengths of the hydrophilic and hydrophobic segments during the whole aggregation process driven by the progressive increase of temperature. The process has been followed by the changes resulting in the dielectric parameters (the dielectric increment Δε and the relaxation frequency ν(0)) of the whole aqueous solution. The dielectric response of the micelles has been described within the framework of the standard electrokinetic model for charged colloidal particles, and the main characteristic parameters have been evaluated. Subsequent cross-linking of these diblock copolymers by a cationic PEO(x)-b-PAMPTMA(y) polyelectrolyte yields hybrid core-shell-corona systems, with the PNIPAAM hydrophobic blocks collapsed in the core, an interpolyelectrolyte chain complex forming the shell, and the hydrophilic PEO chains as an external corona. In this case too, the dielectric spectra can be appropriately accounted for within the same theoretical framework.

Theoretical study of ionic liquids based on the cholinium cation. Ab initio simulations of their condensed phases

We have explored by means of ab initio molecular dynamics the homologue series of 11 different ionic liquids based on the combination of the cholinium cation with deprotonated amino acid anions. We present a structural analysis of the liquid states of these compounds as revealed by accurate ab initio computations of the forces. We highlight the persistent structural motifs that see the ionic couple as the basic building block of the liquid whereby a strong hydrogen bonding network substantially determines the short range structural behavior of the bulk state. Other minor docking features of the interaction network are also discovered and described. Special cases along the series such as Cysteine and Phenylalanine are discussed in the view of their peculiar properties due to zwitterion formation and additional long-range structural organization.

Is a medium-range order pre-peak possible for ionic liquids without an aliphatic chain?

The combination of amino acids anions with a choline cation gives origin to a new and potentially important class of organic ionic liquids that might represent a viable and bio-compatible alternative with respect to the traditional ones. We present here a combined experimental and theoretical study of a choline–proline ionic liquid, using both large and small angle X-ray diffraction (WAXS–SAXS), and classical and ab initio molecular dynamics calculations, in which we are able to point out for the first time the existence of a low Q peak in the X-ray patterns in the absence of linear or branched alkyl chains. From the calculations, we can obtain theoretical scattering patterns that reproduce very nicely the experimental spectra in all Q ranges, and from detailed analysis of the radial distribution functions (RDFs) and hydrogen bond patterns, we can state that very strong ion pairs are established in the liquid and the observed pre-peak can be ascribed to the interactions between atoms belonging to different ion pairs.

Hyaluronic acid and alginate covalent nanogels by template cross-linking in polyion complex micelle nanoreactors

Hyaluronic acid (HA) and alginate (AL) covalent nanogels cross-linked with l-lysine ethyl ester were prepared by template chemical cross-linking of the polysaccharide in polyion complex micelle (PIC) nanoreactors. By using this method we were able to prepare HA and AL nanogels without organic solvents. PICs were prepared by using poly(ethylene oxide)-block-poly[(3-acrylamidopropyl)-trimethylammonium chloride] (PEO-b-PAMPTMA) or poly[(N-isopropylacrylamide)-block-PAMPTMA] (PNIPAAM-b-PAMPTMA). Only PNIPAAM-b-PAMPTMA block copolymers allowed to prepare PIC with small and controlled size. Short polysaccharide chains (Xn=50 and 63 for AL and HA, respectively, where Xn is the number of monosaccharidic units present in the polysaccharide) where used to optimize PIC formation. The remarkable difference in charge density and rigidity of HA and AL did not have a significant influence on the formation of PICs. PICs with small size (diameter of about 50-80 nm) and low polydispersity were obtained up to 5mg/mL of polymer. After cross-linking with l-lysine ethyl ester, the nanoreactors were dissociated by adding NaCl. The nanogels were easily purified and isolated by dialysis. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, TEM and ζ-potential measurements. The size of the smallest nanogels in solution in the swollen state was 50-70 nm in presence of salt and 80-100 nm in water.

Micro- and mesoscopic structural features of a bio-based choline-amino acid ionic liquid

High energy X-ray diffraction data from a bio-based ionic liquid constituted by choline ([cho]) and an amino acid (AA), namely n-leucine ([nle]), are presented and described by means of an atomistic molecular dynamics simulation aiming at extracting detailed structural information at both microscopic and mesoscopic spatial scales. We find that similar to other previously studied analogous systems, a strong, hydrogen bonding driven, cation–anion correlation determines the microscopic structure. While other peculiar correlations exist in this [cho][nle] IL, the medium length alkyl tail of the AA leads to the development of a characteristic polar vs. apolar alternation, as a consequence of the alkyl tails segregation into domains, thus delivering the formation of an enhanced level of mesoscopic organization in this IL.

Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

Self-assembly and drug release study of linear l,d-oligopeptide-poly(ethylene glycol) conjugates.

The preparation and structural organisation of new bioinspired nanomaterials based on regular alternating enantiomeric sequence of tetra- and hexapeptides end-linked to poly(ethylene glycol) (PEG) is reported. The peptide moiety is composed of two or three repeats of l-Ala-d-Val units while the PEG has a molecular weight of 2kDa. The self-assembling properties of the two conjugates depend significantly on the length of the peptide. Nanoparticles with different sizes and morphologies are formed, the structural properties of which are compared with the previously studied l-Ala-d-Val octapeptide conjugate that self-assembles into rod-like nanoparticles. The aggregation properties were studied by NMR, circular dichroism, fluorescence spectroscopies and dynamic light scattering. The morphology and size of the nanoparticles were assessed by scanning electron microscopy and dynamic light scattering. The loading and release of a model drug were also investigated. This study demonstrates that, by changing the length of the peptide, it is possible to modulate the self-assembly and loading properties of peptide-PEG conjugates.

Carbon monoxide binding to the heme group at the dimeric interface modulates structure and copper accessibility in the Cu,Zn superoxide dismutase from Haemophilus ducreyi: in silico and in vitro evidences

X-ray absorption near-edge structure (XANES) spectroscopy and molecular dynamics (MD) simulations have been jointly applied to the study of the Cu,Zn superoxide dismutase from Haemophilus ducreyi (HdSOD) in interaction with the carbon monoxide molecule. The configurational flexibility of the Fe(II)-heme group, intercalated between the two subunits, has been sampled by MD simulations and included in the XANES data analysis without optimization in the structural parameter space. Our results provide an interpretation of the observed discrepancy in the Fe-heme distances as detected by extended X-ray absorption fine structure (EXAFS) spectroscopy and the classical XANES analysis, in which the structural parameters are optimized in a unique structure. Moreover, binding of the CO molecule to the heme induces a long range effect on the Cu,Zn active site, as evidenced by both MD simulations and in vitro experiments. MD simulation of the CO bound system, in fact, highlighted a structural rearrangement of the protein–protein hydrogen bond network in the region of the Cu,Zn active site, correlated with an increase in water accessibility at short distance from the copper atom. In line, in vitro experiments evidenced an increase of copper accessibility to a chelating agent when the CO molecule binds to the heme group, as compared to a heme deprived HdSOD. Altogether, our results support the hypothesis that the HdSOD is a heme-sensor protein, in which binding to small gaseous molecules modulates the enzyme superoxide activity as an adaptive response to the bacterial environment.

Peptides with regularly alternating enantiomeric sequence: From ion channel models to bioinspired nanotechnological applications

Peptides are versatile building blocks that have been extensively used as peptide‐based organizers to generate bioinspired hybrid materials. Among them, those peptides characterized by regularly alternating enantiomeric sequences (l,d‐peptides) have attracted much interest. Their structures, which are not accessible to the corresponding homochiral peptides, have been exploited to achieve hybrid conjugates, the chemical and structural properties of which can be predetermined by correct design. Molecules that self‐assemble into hollow tubular architectures with side chains on the outer surface can form, allowing the introduction of new functionalities on amino acid residues containing reactive side chains. This provides an effective strategy to develop engineered nanotubes with the surface suitably tuned for applications in different fields, spanning from electronics to medicine.